Method of screening Baff suppressor or inhibitor

ABSTRACT

A method of screening a novel BAFF suppressor or inhibitor. More specifically speaking, a method which comprises adding a combination of TPA with ionomycin and/or an anti-CD3 antibody to a cultured human cell to thereby induce the production of BAFF by the cell; a method of screening a substance capable of suppressing the expression or activity of BAFF which comprises adding a test substance to a BAFF-production system prepared by adding a combination of TPA with ionomycin and/or an anti-CD3 antibody to a cultured human cell and measuring the expression amount and/or the activity of BAFF in the BAFF-production system; and a BAFF production inducer for a BAFF-producing cell which contains a combination of TPA with ionomycin and/or an anti-CD3 antibody.

TECHNICAL FIELD

The present invention relates to a method of screening a novel BAFFsuppressor or inhibitor. The present invention relates particularly to amethod of screening a substance suppressing or inhibiting the expressionor production of BAFF by constructing a system wherein a detectablesufficient amount of BAFF is stably released extracellularly bysubjecting a commercial cell strain to suitable stimulating conditions.Specifically, the present invention relates to a method of inducing theproduction of BAFF in a cultured human T cell by adding a combination ofTPA with ionomycin and/or an anti-CD3 antibody to the cell, a method ofscreening a substance capable of regulating the expression or activityof BAFF, which includes adding a test substance to a BAFF productionsystem in which a combination of TPA with ionomycin and/or an anti-CD3antibody are/is added to a cultured human T cell and then measuring theexpression level and/or activity level of BAFF in the BAFF productionsystem, and a BAFF production inducer for a BAFF-producing cell, whichcomprises a combination of TPA with ionomycin and/or an anti-CD3antibody.

BACKGROUND ART

It is known that BAFF (B cell activating factor belonging to the TNFfamily) is produced and secreted from T cells, monocytes/macrophages,dendritic cells and the like and regulates such as B-celldifferentiation, activation, survival rate via 3 types of receptors on Bcells (Moore et al., Science, 285, 260-263 (1999)).

Human BAFF is a transmembrane form protein comprising 285 amino acids.There is a structural characteristic of trimer formation such as thepresence of a cytoplasmic domain of 46 amino acids, an extracellulardomain of 218 amino acids, and two N-glycosylation sites in its aminoacid sequence. It is estimated that an extracellular domain of 152 aminoacids from C-terminal is cleaved with a protease of Furin family andreleased in a soluble form. The amino acid sequence of human BAFFinitially named neutrokine α was disclosed as SEQ ID NO: 1 or 2 inpublication of International Patent Application WO98/18921. Other namesof human BAFF such as Kay, TNFSF13B, Blys, TALL-1, THANK and zTNF4 arealso known.

BAFF-R, TACI (transmembrane activator and calcium modulator andcyclophilin ligand interactor), and BCMA (B cell maturation antigen) areknown as BAFF receptors. BAFF-R and BCMA are expressed mainly in Bcells, and TACI is expressed in B cells and activated T cells.

The physiological action of BAFF lies in regulation of B-celldifferentiation, activation, survival rate and the like as describedabove, and is increasingly revealed in recent years to participate inpathologic condition. That is, it is reported that a mouse expressingBAFF in excess shows SLE-like symptoms such as increase in peripheralblood B cells, enlargement of lymph nodes and spleen, increase in IgGlevel in serum, antinuclear antibody production, deposition of immunecomplex in the kidney, albuminuria and nephritis (Mackay et al., J. Exp.Med., 190, 1697-1710, (1999), and Khare et al., Proc. Natl. Acad. Sci.USA 97, 3370-3375, (2000)). It was further reveled that this mouse alsoshows SS-like symptoms such as inflammation of salivary gland anddestruction of salivary gland with advancing age (Groom et al., J. Clin.Invest., 109, 59-68, (2002)) An increase of BAFF level in serum inpatients suffering from SLE, RA and SS is also reported (Zhanget al., J.Immunol., 166, 6-10, (2001); Cheema et al., Arthritis Rheum., 44,1313-1319, (2001); and Groom et al., J. Clin. Invest., 109, 59-68,(2002)), and there are also many reports such as higher BAFF level insynovial fluid than in serum in patients suffering from RA (Cheema etal., Arthritis Rheum., 44, 1313-1319, (2001)), expression of BAFF insalivary gland-infiltrating leukocytes in patients suffering from SS(Groom et al., J. Clin. Invest. 109, 59-68, (2002)), correlation betweenserum BAFF level in patients suffering from SLE and immunoglobulin oranti-ds DNA antibody (Zhang et al., J. Immunol. 166, 6-10, (2001)) andcorrelation between BAFF in patients suffering from RA and rheumatoidfactor (Cheema et al., Arthritis Rheum., 44, 1313-1319, (2001)).

From these facts, it can be said that the suppression or inhibition ofexpression or production of BAFF leads to prevention and treatment ofautoimmune diseases such as SLE, RA and SS, and thus there is demand forestablishment of a system for accurately screening the suppression orinhibition of expression or production of BAFF.

For establishment of such screening system, it is possible toanticipate, for example, a method of obtaining a cell expressing BAFFstably by transforming a cultured cell with a gene vector constructed soas to express BAFF by recombinant DNA techniques. There is however aproblem that even if BAFF could be expressed, BAFF would not alwaysreleased extracellularly in a secretory form and would be uncertain asto whether the cultured cell can be a cell strain stable for expressionand production of BAFF. Accordingly, none of such system has beenreported. For example, if it would be possible to construct a systemwherein the induction of intracellular expression of BAFF and theextracellular release of a detectable sufficient amount of BAFF can berecognized when a commercial cell strain is subjected to suitablestimulating conditions, the problem described above could be solved.

DISCLOSURE OF INVENTION

The object of the present invention is to provide a method of screeninga substance suppressing or inhibiting the expression or production ofBAFF by constructing a system wherein a detectable sufficient amount ofBAFF is stably released extracellularly by subjecting a commercial cellstrain to suitable stimulating conditions. By constructing such system,there can also be provided a method of screening aprophylactic/therapeutic agent for autoimmune diseases such as systemiclupus erythematosus (SLE), chronic rheumatoid arthritis (RA), Sjogren'ssyndrome (SS), autoimmune diabetes, AIDS, and an autoimmune diseaseaccompanied by B-cell activation.

Accordingly, the present inventors made extensive study on selection ofa cell strain and a method of stimulating the cell, and as a result,found that when a Loucy cell or Jurkat cell that is a commercial human Tcell strain highly expressing CD3 is stimulated with an anti-CD3antibody and/or a combination of TPA (12-O-tetradecanoylphorbol13-acetate, or Phorbol 12-myristate 13-acetate, also expressed as PMA)with ionomycin, the induction of stable BAFF expression in the cell isrecognized and simultaneously a detectable sufficient amount of BAFF canbe stably released into a culture, and the present invention was therebycompleted.

That is, the present invention provides a BAFF production system whereina cultured human T cell is stimulated by adding a combination of TPAwith ionomycin and/or an anti-CD3 antibody to the cell. Specifically,the present invention provides a BAFF production system wherein acultured human T cell such as Loucy cell or Jurkat cell is stimulated byadding a combination of TPA with ionomycin and/or an anti-CD3 antibodyto the cell.

The present invention also relates to a method of inducing theproduction of BAFF in a cultured human T cell, by adding a combinationof TPA with ionomycin and/or an anti-CD3 antibody to the cell.Specifically, the present invention relates to a method of inducing theproduction of BAFF in a cultured human T cell such as Loucy cell orJurkat cell, by adding a combination of TPA with ionomycin and/or ananti-CD3 antibody to the cell.

The present invention also provides a method of screening a substancecapable of regulating the expression or activity of BAFF in a BAFFproduction system wherein a cultured human T cell is stimulated byadding a combination of TPA with ionomycin and/or an anti-CD3 antibodyto the cell. Specifically, the present invention provides a method ofscreening a substance capable of suppressing or inhibiting theexpression or activity of BAFF in a BAFF production system wherein acultured human T cell is stimulated by adding a combination of TPA withionomycin and/or an anti-CD3 antibody to the cell. More specifically,the present invention provides a method of screening a substance capableof regulating, preferably suppressing or inhibiting the expression oractivity of BAFF in a BAFF production system wherein a cultured human Tcell such as Loucy cell or Jurkat cell is stimulated by adding acombination of TPA with ionomycin and/or an anti-CD3 antibody to thecell.

The screening method of the present invention relates particularly to amethod of screening a substance capable of regulating the expression oractivity of BAFF, which includes adding a test substance to a BAFFproduction system in which a combination of TPA with ionomycin and/or ananti-CD3 antibody are/is added to a cultured human T cell and thenmeasuring the expression level and/or activity level of BAFF in the BAFFproduction system. Specifically, the present invention relates to amethod of screening a substance capable of regulating the expression oractivity of BAFF, which includes adding a test substance to a BAFFproduction system in which a combination of TPA with ionomycin and/or ananti-CD3 antibody are/is added to a cultured human T cell such as Loucycell or Jurkat cell and then measuring the expression level and/oractivity level of BAFF in the BAFF production system. More specifically,the present invention relates to a method of screening a substancecapable of suppressing or inhibiting the expression or activity of BAFF,which includes adding a test substance to a BAFF production system inwhich a combination of TPA with ionomycin and/or an anti-CD3 antibodyare/is added to a cultured human T cell such as Loucy cell or Jurkatcell and then measuring the expression level and/or activity level ofBAFF in the BAFF production system.

Further, the present invention provides a method of screening aprophylactic/therapeutic agent for autoimmune diseases such as systemiclupus erythematosus (SLE), chronic rheumatoid arthritis (RA), Sjogren'ssyndrome (SS), autoimmune diabetes, AIDS, and an autoimmune diseaseaccompanied by B-cell activation, in a BAFF production system wherein acultured human T cell is stimulated by adding a combination of TPA withionomycin and/or an anti-CD3 antibody to the cell. Specifically, thepresent invention provides a method of screening aprophylactic/therapeutic agent for autoimmune diseases such as systemiclupus erythematosus (SLE), chronic rheumatoid arthritis (RA), Sjogren'ssyndrome (SS), autoimmune diabetes, AIDS, and an autoimmune diseaseaccompanied by B-cell activation, in a BAFF production system wherein acultured human T cell such as Loucy cell or Jurkat cell is stimulated byadding an anti-CD3 antibody and/or a combination of TPA with ionomycinto the cell. More specifically, the present invention provides a methodof screening a prophylactic/therapeutic agent for autoimmune diseasessuch as systemic lupus erythematosus (SLE), chronic rheumatoid arthritis(RA), Sjogren's syndrome (SS), autoimmune diabetes, AIDS, and anautoimmune disease accompanied by B-cell activation, which includesadding a test substance to a BAFF production system in which acombination of TPA with ionomycin and/or an anti-CD3 antibody are/isadded to a cultured human T cell such as Loucy cell or Jurkat cell andthen measuring the expression level and/or activity of BAFF in the BAFFproduction system.

Further, the present invention relates to a BAFF production inducer fora BAFF-producing cell, which contains a combination of TPA withionomycin and/or an anti-CD3 antibody. Specifically, the presentinvention relates to a BAFF production inducer for a cultured human Tcell, which contains a combination of TPA with ionomycin and/or ananti-CD3 antibody. More specifically, the present invention relates to aBAFF production inducer for a cultured human T cell such as Loucy cellor Jurkat cell, which contains a combination of TPA with ionomycinand/or an anti-CD3 antibody.

According to the present invention, there can be provided a method ofscreening, with a cultured cell strain, a substance capable ofsuppressing or inhibiting the production of BAFF as well as a method ofscreening a prophylactic/therapeutic agent for diseases involved inproduction or activity of BAFF, such as systemic lupus erythematosus(SLE), chronic rheumatoid arthritis (RA), Sjogren's syndrome (SS),autoimmune diabetes, AIDS, and an autoimmune disease accompanied byB-cell activation.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a drawing showing the expression of BAFF upon stimulation of aLoucy cell or Jurkat cell with an anti-CD3 antibody or both TPA andionomycin.

FIG. 2 is a drawing showing the expression of BAFF protein uponstimulation of a Loucy cell with an anti-CD3 antibody or both TPA andionomycin.

FIG. 3 is a drawing in which recombinant human BAFF (manufactured byChemicon) was detected by conventional Western blotting method with anantihuman BAFF monoclonal antibody (4H4) and a control rabbit antihumanBAFF polyclonal antibody (Chem) manufactured by Chemicon.

FIG. 4 is a graph showing the production of BAFF upon stimulation of aLoucy cell with TPA and ionomycin.

FIG. 5 is a graph showing the production of BAFF upon stimulation of aJurkat cell with TPA and ionomycin.

BEST MODE FOR CARRYING OUT THE INVENTION

The inventors examined the expression of BAFF upon stimulation of aLoucy cell or Jurkat cell with various substances. As a result, theinventors found that when a Loucy cell or Jurkat cell is stimulated withan anti-CD3 antibody or a combination of TPA with ionomycin, theinduction of stable BAFF expression in the cell is recognized andsimultaneously a detectable sufficient amount of BAFF can be stablyreleased into a culture, as shown later in the Examples.

Accordingly, the method of screening a substance capable of regulating,preferably suppressing or inhibiting the expression or activity of BAFFcan be constituted as follows.

That is, commercial human T cells are suspended at a suitable density ina suitable medium, then seeded in a culture plate or the like, andstimulated by adding TPA (final concentration of 0.001 to 1.0 μg/mL,preferably 0.005 to 0.1 μg/mL) and ionomycin (final concentration of0.01 to 10.0 μg/mL, preferably 0.05 to 1.0 μg/mL), and a test substance(any concentration gradients at a final concentration of 10⁻¹⁵ to 10⁻³M) is added as necessary, and culture of the cells is initiated. Theculture supernatant is recovered 1 hour to 7 days, preferably 3 hours to5 days, after the culture is initiated, and the concentration of BAFF inthe supernatant can be measured and compared with the control, therebymeasuring the activity of the test substance to suppress or inhibit BAFFin this screening system. For carrying out the above-described system, aculture plate coated previously with an anti-CD3 antibody diluted withPBS or the like (final concentration of 0.01 to 1000 μg/mL, preferably0.1 to 100 μg/mL) may be used. The culture plate or the like coated withan anti-CD3 antibody can be used to detect a sufficient amount of BAFFwithout using TPA and ionomycin and is useful as an easier screeningsystem.

The human T cell strain used in the present invention is preferably acommercial cell, and examples include a Loucy cell (ATCC Cat. No.CRL-2629), a Jurkat cell (ATCC Cat. No. TIB-152) and a TALL-1 cell (JCRB0086), among which the Loucy cell (ATCC Cat. No. CRL-2629) or Jurkatcell (ATCC Cat. No. TIB-152) highly expressing CD3 and expected to beactivated via a T-cell receptor is more preferable.

The anti-CD3 antibody used in the present invention includes anantihuman CD3 monoclonal antibody and antihuman CD3 polyclonal antibodyproduced by methods known by those skilled in the art, among which anantihuman CD3 monoclonal antibody is preferable, and a mouse antihumanCD3 monoclonal antibody (BD Pharmingen Cat No. 555336, or the like) ismore preferable.

TPA used in the present invention, which is an abbreviation of12-O-tetradecanoylphorbol 13-acetate, is a phorbol ester also known asphorbol 12-myristate 13-acetate (abbreviated sometimes as PMA), and TPAused in the present invention may be any of compounds given such namesand also includes stereoisomers. Preferable commercial TPA includes, forexample, 4-α-phorbol 12-myristate 13-acetate (SIGMA Cat. No. P-148).

Ionomycin used in the present invention is a polyether antibioticderived from Streptomyces conglobatus and is known to act as calciumionophore, and ionomycin used in the present invention also includespharmaceutically acceptable salts. Preferable commercially availableionomycin includes, for example, ionomycin, calcium salt (SIGMA Cat. No.IO634).

There is a characteristic that BAFF production inducer of the presentinvention contains a combination of TPA with ionomycin and/or ananti-CD3 antibody as active ingredients for inducing the production ofBAFF. The BAFF production inducer of the present invention can comprise,in addition to the above active ingredients, a carrier such as a buffersolution which can be added to a cell system, and together with thecarrier which can be added to a cell system, can be formed into acomposition for inducing the production of BAFF.

The BAFF production inducer of the present invention can be applied to aBAFF-producing cell, preferably a cultured cell, and preferable examplesof the cell include a cultured human T cell, specifically a Loucy celland Jurkat cell.

When the BAFF production inducer of the present invention includes acombination of TPA with ionomycin as the active ingredients, therespective active ingredients are used by adding them to a cell systemin such amounts that the final concentration of TPA becomes 0.001 to 1.0μg/mL, preferably 0.005 to 0.1 μg/mL, while the final concentration ofionomycin becomes 0.01 μg/mL to 10 μg/mL, preferably 0.05 μg/mL to 1μg/mL. When the active ingredient is an anti-CD3 antibody, the activeingredient can be put to a culture plate at a concentration of 0.1 μg/mLto 100 μg/mL, preferably 1 μg/mL to 30 μg/mL in a buffer solution.

The thus obtained BAFF production system of the present invention has asurprising feature that a detectable sufficient amount of BAFF can bestably released into a culture as shown later in the Examples, and thusit is possible to realize not only a method of screening a substancecapable of regulating, preferably suppressing or inhibiting theexpression or activity of BAFF, but also a method of screening aprophylactic/therapeutic agent for autoimmune diseases such as systemiclupus erythematosus (SLE), chronic rheumatoid arthritis (RA), Sjogren'ssyndrome (SS), autoimmune diabetes, AIDS, and an autoimmune diseaseaccompanied by B-cell activation.

EXAMPLES

Hereinafter, the present invention is described in more detail byreference to the Examples, but the technical scope of the presentinvention is not limited by the Examples.

Example 1 Expression of BAFF upon stimulation of a Loucy cell or JurkatCell with an Anti-CD3 Antibody or Both TPA and Ionomycin

(a) Culture Under Stimulation with TPA and Ionomycin

Loucy cells or Jurkat cells purchased from ATCC (American Type CultureCollection) were suspended in RPMI1640 medium containing 10% FBS (Fetalbovine serum), 0.45% D-glucose, 1 mM sodium pyruvate and 1 mM HEPES andseeded at a density of 1×10⁶ cells/well on a 6-well culture plate. Atthe same time, TPA and ionomycin were added respectively to a finalconcentration of 0.05 μg/mL, 0.5 μg/mL or 0.1 μg/mL, and the cells wererecovered 5 hours, 24 hours, 48 hours and 72 hours (Loucy cells) or 48hours, 120 hours and 168 hours (Jurkat cells) after the culture wasinitiated. Total RNA was extracted from these recovered cells, and BAFFmRNA was analyzed by the RT-PCR method under the following conditions.Using ReverTra Dash kit (TOYOBO Cat. No. PCR-401), the sample wassubjected to reverse transcription reaction at 30° C. for 10 minutes, at42° C. for 20 minutes and at 99° C. for 5 minutes to give cDNA. UsingAdvantage 2 PCR Enzyme system (BD Biosciences Cat. No. PT3281-1), thiscDNA was reacted at 94° C. for 1 minute, then subjected to 35 reactioncycles each at 94° C. for 30 seconds and at 66° C. for 1.5 minutes andreacted at 66° C. for 5 minutes, and the DNA thus acquired was separatedby 1.5% agarose gel electrophoresis, and an objective band wasrecognized at a position of 975 bp. The primer sequences used in the PCRreaction are as follows:

Forward: 5′aggccccaaccttcaaagttcaag (SEQ ID NO: 1) Reverse:5′-cttagaggtacagagaaagggagg (SEQ ID NO: 2)

The results are shown in FIG. 1. FIG. 1( a) shows the sample from theLoucy cells, and FIG. 1( b) shows the sample from the Jurkat cells. T+Ishows stimulation with TPA and ionomycin (which are 0.05 μg/mL and 0.5μg/mL respectively in (a); and 0.05 μg/mL and 0.5 μg/mL (on theobservers' right) or 0.1 μg/mL (on the observers' left) in (b)), and (−)shows non-stimulation. In each profile, G3PDH is a positive control. InLoucy cells and Jurkat cells stimulated with TPA and ionomycin, theexpression of BAFF mRNA was recognized 5 hours and 2 days after thestimulation, and the induction of evident expression of BAFF mRNA by TPAand ionomycin was continuously recognized over 72 hours and 7 days (168hours) after the culture was initiated, as shown in FIGS. 1( a) and (b).It was thus found that because the induction of stable expression ofBAFF was recognized by culturing Loucy cells or Jurkat cells understimulation with TPA and ionomycin, this culture system can be used inscreening a substance suppressing or inhibiting the expression of BAFF.

(b) Culture Under Stimulation with an Anti-CD3 Antibody

An anti-CD3 antibody diluted at 10 μg/mL with PBS was put to a 6-wellplate and adsorbed onto the bottom at 4° C. overnight, and then Loucycells suspended in the same manner as in (a) were seeded at a density of1×10⁶ cells/well. The cells were recovered 5 hours, 24 hours, 48 hoursand 72 hours after the culture was initiated. Total RNA was extractedfrom these recovered cells, and BAFF mRNA was analyzed by the RT-PCRmethod under the following conditions. Using ReverTra Dash kit (TOYOBOCat. No. PCR-401), the sample was subjected to reverse transcriptionreaction at 30° C. for 10 minutes, at 42° C. for 20 minutes and at 99°C. for 5 minutes to give cDNA. Using Advantage 2 PCR Enzyme system (BDBiosciences Cat. No. PT3281-1), this cDNA was reacted at 94° C. for 1minute, then subjected to 35 reaction cycles each at 94° C. for 30seconds and at 66° C. for 1.5 minutes and reacted at 66° C. for 5minutes, and the acquired DNA was separated by 1.5% agarose gelelectrophoresis, and an objective band was recognized at a position of975 bp. As a result, two-phase expression was made evident as shown inanti-CD3 in the right in FIG. 1( a), wherein the expression of BAFF mRNAis recognized from 5 hours after stimulation, thereafter the expressionis attenuated, and the mRNA is expressed again 72 hours after theculture is initiated. There would be a possibility that such expressionmodes are different in biological meaning, and it is considered that theexpression of BAFF induced by culturing Loucy cells under stimulationwith an anti-CD3 antibody can be used in screening a substancesuppressing or inhibiting the unique expression of BAFF.

Example 2 Expression of BAFF Protein Upon Stimulation of a Loucy Cellwith an Anti-CD3 Antibody or Both TPA and Ionomycin

An anti-CD3 antibody diluted at 10 μg/mL with PBS was put to a 6-wellplate and adsorbed onto the bottom at 4° C. overnight, and then Loucycells suspended in the same manner as in Example 1 were seeded at adensity of 1×10⁶ cells/well. The cells were recovered 24 hours, 48hours, 72 hours and 96 hours after the culture was initiated. Theserecovered cells were used and examined for expression of BAFF protein bya conventional Western blotting method. As an antibody for detection ofBAFF protein, a rabbit antihuman BAFF polyclonal antibody (AB16530,manufactured by Chemicon) was used. The results are shown in FIG. 2. Acontrol not stimulated is shown in (−) in the left in FIG. 2, and CD3stimulation in FIG. 2 shows stimulation with anti-CD3 antibody, andTPA+Ionomycin shows stimulation with TPA and ionomycin. β-actin in thelower profile in FIG. 2 shows a positive control. In the Loucy cellsstimulated with an anti-CD3 antibody, a 34-kDa band corresponding tohuman BAFF was confirmed from 24 hours after stimulation, and thisevident expression induction was continuously recognized over 96 hoursafter the culture was initiated. Separately, Loucy cells were suspendedin RPMI1640 medium containing 10% FBS (fetal bovine serum), 0.45%D-glucose, 1 mM sodium pyruvate and 1 mM HEPES and seeded at a densityof 1×10⁶ cells/well on a 6-well culture plate, and at the same time,simultaneously TPA and ionomycin were added to final concentrations of0.05 μg/mL and 0.5 μg/mL respectively, and the cells were recovered 24hours, 48 hours, 72 hours and 96 hours after the culture was initiated.These recovered cells were used and examined for expression of BAFFprotein by a conventional Western blotting method. In the Loucy cellsstimulated with TPA and ionomycin, a 34-kDa band corresponding to humanBAFF was recognized from 24 hours after stimulation, and this evidentexpression induction was continuously recognized over 96 hours after theculture was initiated, as shown in FIG. 2. In these results, theinduction of stable expression of BAFF protein by culturing Loucy cellsunder stimulation with an anti-CD3 antibody or both TPA and ionomycinwas recognized over 96 hours after the culture was initiated, and thisculture system can be used in screening a substance suppressing orinhibiting the expression of BAFF protein.

Reference Example 1 Preparation of Antihuman BAFF Monoclonal Antibody(4H4)

The antihuman BAFF monoclonal antibody (4H4) used in Example 3 wasprepared in the following manner. That is, 13 amino acids (SEQ ID NO: 4)corresponding to a region, in the vicinity of a membrane, of anextracellular domain in 285 amino acids of BAFF shown in SEQ NO: 3 inthe Sequence Listing were selected, then conjugated with KLH by the MBSmethod, and used as antigen. 100 μL of 1 mg/ml aqueous solution of theantigen peptide in physiological saline and Freund's complete adjuvantwere formed into an emulsion and then used in intraperitoneallyimmunizing a mouse (Balb/c, 6-week-old). After 2 weeks, 100 μL of 1mg/ml of an aqueous solution of the antigen peptide in physiologicalsaline and Freund's complete adjuvant, which had been emulsified bysonication, were used as booster for additional immunization, followedby additional immunization twice at 2-week intervals. Two months afterthe first immunization, the spleen was excised, and lymphocytes wereseparated in RPMI 1640 medium (containing penicillin and streptomycin).The separated lymphocytes were fused with mouse bone marrow-derivedmyeloma cell P3U1 strain by the polyethylene glycol (PEG) method toprepare hybridoma cells. The hybridoma cells were suspended in a feedercell-containing HAT medium, pipetted to a 96-well plate (Greiner) andcultured for 15 days. A culture supernatant was recovered from the wellsin which the hybridoma cells had been cultured, and antibody-producingcells reactive with the antigen peptide were selected by ELISA(enzyme-linked immunosorbent assay). That is, first, 50 μL of 10 μg/mLantigen peptide was put to each well of the 96-well plate, adsorbed ontothe bottom at 4° C. overnight and blocked with 100 μL of 2% BSA/PBS at37° C. for 2 hours. Each well was reacted at 4° C. overnight with 100 μlsupernatant of the hybridoma cells and then reacted at 37° C. for 1 hourwith a 1000-fold dilution of HRP-labeled anti-mouse IgG and colored withorthophenylene diamine as substrate. After the reaction was terminatedwith 50 μL of 2 N sulfuric acid, each well was measured for absorptionat 492 nm, and hybridomas showing an absorption of 1.0 or more wereselected and cloned by limiting dilution.

The selected hybridoma cells were injected intraperitoneally to a mouse(Balb/c) to which 0.5 mL pristane had been intraperitoneallyadministered before 7 days and before 3 days, and about 10 days later,the ascites fluid was collected. The recovered ascites fluid was left atroom temperature for 30 minutes, then left at 4° C. overnight,centrifuged at 15 Krpm for 10 minutes to recover a supernatant fromwhich a mouse IgG fraction was separated and purified through a proteinA-Sepharose column.

By the method described above, a hybridoma cell strain producing theantihuman BAFF antibody (4H4 whose isotype is IgG1), as well as theantibody (4H4), was obtained. The resulting 4H4, and a control rabbitantihuman BAFF polyclonal antibody (AB16530: shown as Chem in FIG. 3)manufactured by Chemicon, were used in detection of recombinant humanBAFF (manufactured by chemicon) by the conventional Western blottingmethod. As a result, a 17-KDa band corresponding to the soluble humanBAFF was confirmed as shown in FIG. 3.

Example 3 Production of BAFF Upon Stimulation of a Loucy Cell or JurkatCell with TPA and Ionomycin

Loucy cells or Jurkat cells suspended in the same manner as in Example 1were seeded to a 6-well culture plate at a density of 1×10⁶ cells/well.At the same time, TPA and ionomycin were added to final concentrationsof 0.05 μg/mL and 0.5 μg/mL respectively, and the culture supernatantwas recovered 5 hours, 24 hours, 48 hours, 72 hours (Loucy cells) or 1,4 and 7 days (Jurkat cells) after the culture was initiated. BAFFproduced by the cells contained in these culture supernatants wasmeasured by an ELISA method using a commercial rabbit antihuman BAFFpolyclonal antibody (Cat No. AB16530, manufactured by Chemicon) and themouse antihuman BAFF monoclonal antibody (4H4) prepared in ReferenceExample 1. The ELISA method was carried out in the following manner.That is, a 96-well plate was coated at 4° C. overnight with 1 μg/wellrabbit antihuman BAFF polyclonal antibody (Cat. No. AB16530,manufactured by Chemicon) as primary antibody. Each well was washed 3times with PBS containing 0.05% Tween 20, and then Block Ace (DainipponPharmaceutical Co., Ltd.) was added in a volume of 150 μL/well andreacted at 37° C. for 2 hours. Each well was washed 3 times with PBScontaining 0.05% Tween 20, and 50 μL sample and 50 μl (8 ng/mL) ofbiotin-labeled 4H4 were added and reacted at room temperature for 2hours. Each well was washed 3 times with PBS containing 0.05% Tween 20,and 50 μL of a 1000-fold dilution of streptoavidin-labeled HRP (HorseRadish peroxide) diluted with PBS containing 0.05% Tween 20 was addedand reacted at room temperature for 30 minutes. Each well was washed 5times with PBS containing 0.05% Tween 20, and then 50 μL of TMB OneSolution (manufactured by Clonetech) was added and reacted for 5 minutesat room temperature, then 50 μL of 1 N HCl was added, and the each wellwas measured for absorbance at 450 nm with a plate reader (manufacturedby Perkin Elmer). As a standard substance, recombinant human BAFF (CatNo. GF119, manufactured by Chemicon) was used to prepare a standardcurve, from which the concentration of BAFF in the sample was measured.

A graph showing the result where the Loucy cells were used is shown inFIG. 4, and a graph showing the result where the Jurkat cells were usedis shown in FIG. 5. The concentration of BAFF (ng/mL) is shown on theordinate in each graph, and each left bar graph shows, as control, theconcentration of BAFF without stimulation with TPA and ionomycin. Theresults indicated, as shown in FIGS. 4 and 5, that in the Loucy cells orJurkat cells stimulated with TPA and ionomycin, BAFF in the culturesupernatant can be detected from 5 hours or 1 day after stimulation andis continuously detected over 72 hours or 7 days after the culture wasinitiated. From these results, it was revealed that the induction ofstable expression of BAFF was recognized by culturing Loucy cells orJurkat cells under stimulation with TPA and ionomycin, and this culturesystem can be used in screening a substance suppressing or inhibitingthe production of BAFF.

INDUSTRIAL APPLICABILITY

The present invention provides a method of inducing, in a cultured humanT cell, the production of BAFF revealed to be related to autoimmunediseases such as systemic lupus erythematosus (SLE), chronic rheumatoidarthritis (RA), Sjogren's syndrome (SS), autoimmune diabetes, AIDS, andan autoimmune disease accompanied by B-cell activation, and provides atechnique of screening a new substance useful for treatment andprevention of autoimmune diseases, and is industrially applicable.

1. A method of screening a substance capable of regulating theexpression or activity of B cell activating factor belonging to thetumor necrosis factor family (BAFF), comprising: adding a test substanceto a BAFF production system including a Loucy cell stimulated by acombination of 12-O-tetradecanoylphorbol 13-acetate (TPA) and ionomycin,and then measuring at least one of the expression level and activitylevel of BAFF in the BAFF production system.
 2. The method according toclaim 1, wherein the regulation of expression or activity of BAFF is thesuppression or inhibition of expression or activity of BAFF.